Vascular endothelial growth factor (VEGF) has many important effects on the vasculature, including vasodilation, increased capillary permeability, vasculogenesis, and angiogenesis (1). Many of these biological actions of VEGF involve signaling mediated by nitric oxide (NO) (2), likely both by induction of gene expression and by enzymatic activation of endothelial NO synthase (eNOS) (3). Activation of eNOS by VEGF is mediated through several pathways, including an increase in intracellular concentrations of Ca2+([Ca2+]i) and by phosphorylation of several residues of eNOS. Protein kinase Akt can directly phosphorylates eNOS on Ser1179 (the bovine homologue of human Ser1177), and this residue is considered one of the most important for regulation of eNOS activity by phosphorylation (4). Activation of eNOS by increased [Ca2+]i, is quick and lasts only a few minutes, whereas eNOS Ser1179 phosphorylation results in more sustained activation, lasting half an hour or longer (5). Certain growth factors and hormones, including VEGF, activate eNOS by both mechanisms (6). In contrast, activation of eNOS by insulin is largely dependent on eNOS Ser1179 phosphorylation and independent of [Ca2+]i (6, 7).
Certain signaling pathways activated by VEGF is mediated by protein kinase C (PKC). VEGF increases PKC activity and PKCβ activation contributes to VEGF-stimulated endothelial cell proliferation (8) and increased vascular permeability (9). Consistent with these observations, inhibitors of PKC block phosphorylation of eNOS Ser1179 (10) and NO production (11) stimulated by VEGF in endothelial cells. Both insulin and VEGF increase eNOS Ser1179 phosphorylation through activation of PI3K and Akt (12, 13). Importantly, PKC activation by phorbol ester decreases insulin-stimulated PI3K activity (14), but inhibition of PI3K does not block PKC activation stimulated by VEGF (8, 15).